(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
21 February 2002 (21.02.2002)
PCT
llllllllllllllllllllllllllllllllll^
(10) International Publication Number
WO 02/14281 Al
(51) International Patent Classification^: C07D 213/75,
401/12, A61K 31/4418, A61P 7/00, 9/00, 17/00, 25/00,
31/00, C07F 9/58
(21) International Application Number: PCT/USO 1/250 15
(22) International Filing Date: 10 August 2001 (10,08.2001)
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, H, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA,
ZW.
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/224,719
1 1 August 2000 (1 1.08.2000) US
(71) Applicants and
(72) Inventors: COCHRAN, John [US/US]; 24 Royal Crest
Drive #3, North Andover, MA 01845 (US). GALULLO,
Vincent [US/US]; 18A Ayer Road, Harvard, MA 01451
(US). BEMIS, Guy [US/US]; 256 Appleton Street, Arling-
ton, MA 02476 (US).
(74) Agents: HALEY, James, F.; Fish & Neave, 1251 Avenue
of the Americas, New York, NY 10020 et al. (US).
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BE, BJ, CF,
CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD,
TG).
Published:
— with international search report
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
00
o
(54) Title: INHIBITORS OF p38
(57) Abstract: The present invention relates to inhibitors of p38, a mammalian protein kinase involved cell proliferation, cell death
and response to extracellular stimuli. The invention also relates to inhibitors of ZAP70. The invention also relates to methods for
producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors of the invention and
methods of utilizing those compositions in the treatment and prevention of various disorders.
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PYRIDINE DERIVATIVES AS INHIBITORS OF P3S
TECHNICAL FIELD OF INVENTION
5
The present invention relates to inhibitors of
p38, a maimnalian protein kinase involved in cell
proliferation, cell death and response to extracellular
stimuli. The invention also relates to methods for
10 producing these inhibitors. The invention also provides
pharmaceutical compositions comprising the inhibitors of
the invention and methods of utilizing those compositions
in the treatment and prevention of various disorders.
BACKGRQIMD OF THE INVENTION
Protein kinases are involved in various
cellular responses to extracellular signals. Recently, a
family of mitogen-activated protein kinases (MAPK) has
been discovered. Members of this family are Ser/Thr
kinases that activate their substrates by phosphorylation
[B. Stein et al . , Ann. Rep. Med. Chem., 31, pp. 289-98
(1996) ] . MAPKs are themselves activated by a variety of
signals including growth factors, cytokines, UV
radiation, and stress-inducing agents.
One particularly interesting MAPK is p38. p38,
also known as cytokine suppressive anti-inflammatory drug
binding protein (CSBP) and RK, was isolated from murine
pre-B cells that were transfected with the
lipopolysaccharide (LPS) receptor, CD14, and induced with
LPS. p38 has since been isolated and sequenced, as has
the cDNA encoding it in humans and mouse. Activation of
15
20
25
30
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p38 has been observed in cells stimulated by stress, such
as treatment of lipopolysaccharides (LPS) , UV,
anisomycin, or osmotic shock, and by cytokines, such as
IL-1 and TNF.
5 Inhibition of p38 kinase leads to a blockade on
the production of both IL-1 and TNF. IL-1 and TNF
stimulate the production of other proinflammatory
cytokines such as IL-6 and IL-8 and have been implicated
in acute and chronic inflammatory diseases and in post-
10 menopausal osteoporosis [R. B. Kimble et al . ,
Endocrinol., 136, pp, 3054-61 (1995)].
Based upon this finding, it is believed that
p38, along with other mPKs, have a role in mediating
cellular response to inflammatory stimuli, such as
15 leukocyte accumulation, macrophage/monocyte activation,
tissue resorption, fever, acute phase responses and
neutrophilia. In addition, MAPKs, such as p38, have been
implicated in cancer, thrombin- induced platelet
aggregation, immunodeficiency disorders, autoimmune
20 diseases, cell death, allergies, osteoporosis and
neurodegenerative disorders. Inhibitors of p38 have also
been implicated in the area of pain management through
inhibition of prostaglandin endoperoxide synthase-2
induction. Other diseases associated with Il-l, IL-6,
25 IL-8 or TNF overproduction are set forth in WO 96/21654.
Others have already begun trying to develop
drugs that specifically inhibit MAPKs. For example, PCT
publication WO 95/31451 describes pyrazole compounds that
inhibit MAPKs, and, in particular, p38. However, the
30 efficacy of these inhibitors in vivo is still being
investigated.
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Other p38 inhibitors have been produced,,
including those described in WO 98/27098, WO 99/00357, WO
99/10291, WO 99/58502, WO 99/54400, WO 00/17175 and WO
00/17204 .
5 Accordingly, there is still a great need to
develop other potent inhibitors of p38, including p38-
specific inhibitors, that are useful in treating various
conditions associated with p38 activation.
Another protein kinase that is involved in
10 cellular responses to extracellular signals is ZAP70.
When the T cell receptor (TOR) in T cells is triggered by
binding an antigen, it in turn activates ZAP7 0. ZAP7 0
acts to couple the TCR to a niimber of essential
signalling pathways that are required for T cell
15 differentiation and proliferation.
Given ZAP70's role in T cell signalling, ZAP70
may have a role in T cell mediated diseases. Such
diseases include, without limitation, transplantation,
autoimune disease, e.g., RA, systemic lupus erythematosus
20 (SLE), psoriasis, Sjogren's Syndrome, thyroiditis,
pulmonary fibrosis, bronchiolitis obliterans, hemolytic
anemia and Wegener's granulomatosis, cancer, including
leukemia and lymphoma, multiple sclerosis, graft versus
host disease, and Kawasaki syndrome.
-5 Accordingly, there is a great need to develop
inhibitors of ZAP70 that are useful in treating various
conditions associated with ZAP70 activation.
SUMMARY OF THE INVENTION
The present invention addresses this problem by
providing compounds that demonstrate inhibition of p38
and/or ZAP70.
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4-
10
These compounds have the general formula
Q2
(la), (lb).
O
R
7
(Ic) and (Id),
wherein each of Qi and Q2 are independently selected from
a phenyl or 5-6 membered aromatic heterocyclic ring
system, or a 8-10 membered bicyclic ring system
comprising aromatic carbocyclic rings, aromatic
heterocyclic rings or a combination of an aromatic
carbocyclic ring and an aromatic heterocyclic ring.
A heterocyclic ring system or a heterocyclic
ring contains 1 to 4 heteroatoms, which are independently
15 selected from N, O, S, SO and SO2 .
The rings that make up Qi are substituted with
1 to 4 substituents, each of which is independently
selected from halo; C1-C3 alkyl optionally substituted
with NR'2, OR', CO2R' or C0NR''2; O- (C1-C3) -alkyl
20 optionally substituted with NR'2/- OR', CO2R' or CONR'2;
NR'2; OCF3; CF3/ NO2/ CO2R'; CONR'; SR'; S(02)N(R')2; SCF3;
CN; N(R')C(0)R\- N (R' ) C (O) ORS' N (R' ) C (O) C (O) rS*
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N(R' ) S (02)RS- N{R')R'; N(R')2/ ORS- 0C(0)R'; 0P(0)3H2; or
N=C-N(R' ) 2.
The rings that make up Q2 are optionally
substituted with up to 4 substituents, each of which is
5 independently selected from halogen; C1-C3 straight or
branched alkyl optionally substituted with R' , NR'2/ OR',
CO2R'. S(02)N(R')2, N=C-N(RM2, R^ 0-P{03)H2, or CONR'2; O-
(C1-C3) -alkyl; O- (C1-C3) -alkyl optionally substituted with
NR'2. OR', CO2R', S(02)N(R^)2. N=CR'-N(R' )2, R^ 0P(O3)H2,
10 or coNR^2; NR'2; 0CF3; CF3; NO2; CO2R' ; coNR'2; R^; or^;
NR^2; SR^; C(0)R^; C(0)N(R')R^; C(0)0R^; SR' ; S{02)N(R')2;
SCF3; N=CR'-N(R' )2; ORS- 0-C02R\' N(RMC(0)R';
N(R' )C(0)0R'; N(R' )C(0)C(0)R'; N (R' ) S {O2) r' ; N(RMR^;
N(R')2; ORS- 0C(0)RS- 0P(0)3H2; K; or CN.
15 Each R' is independently selected from
hydrogen; (C1-C3) -alkyl; (C2-C3) -alkenyl or alkynyl;
phenyl or phenyl substituted with 1 to 3 substituents
independently selected from halo, methoxy, cyano, nitro,
amino, hydroxy, methyl or ethyl; or a 5-6 membered
20 heterocyclic ring system optionally substituted with 1 to
3 substituents independently selected from halo, methoxy,
cyano, nitro, amino, hydroxy, methyl or ethyl.
Each R is independently selected from hydrogen,
-R^ -N(R^)2/ -0R^ SR^ -C(0)-N(R^)2, -S (O2 ) -N (R') 2,
25 -0(0) -OR^ or -C{0)R^ wherein two adjacent R are optionally
bound to one another and, together with each Y to which
they are respectively bound, form a 4-8 membered
carbocyclic or heterocyclic ring.
Each R^ is independently selected from
30 hydrogen; or (C1-C3) -alkyl or (C1-C3) -alkenyl, each
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optionally substituted with -N(R')2/ -OR', SR' , -0-C (O)
N(R')2. -C(0)-N(R' )2, -S (02)-N(R' )2, -C(0)-OR', -NS02R\
NS02R^, -C(0)N(R' ) (R^) , -NC(0)R^ -N(R')(R^), -N(R')(R^),
C(0)R^, -C(0)N(R' ) (R^) , -N(R^)2, -C(0)N=C(NH)2 or R^.
5 Each R is independently selected from 5-8
membered aromatic or non-aromatic carbocyclic or
heterocyclic ring systems each optionally substituted
with R', r\ -C(0)RS -C(0)r\ -C(0)0R^ or -K; or an 8-1
membered bicyclic ring system comprising aromatic
10 carbocyclic rings, aromatic heterocyclic rings or a
combination of an aromatic carbocyclic ring and an
aromatic heterocyclic ring each optionally substituted
with R'^ , r\ -C(0)R', -C{0)r\ -C(0)OR^ or -K.
Each R is independently selected from R'; (Ci
15 C7) -straight or branched alkyl optionally substituted
with RS N{R')2, OR^ , CO2R' , CON(R')2, S02N(R')2or
5
S02N(R )2f or a 5-6 membered carbocyclic or heterocyclic
ring system optionally substituted with N(R')2/ OR',
CO2R' , C0N(R')2/ S02N(RM2or S02N(R^)2.
2 0 Each R^ is independently selected from
hydrogen, (C1-C3) -alkyl, or (C1-C3) -alkenyl; each
optionally substituted with -N(R')2/ -OR', SR' , -C(0)-
N(R')2/ -S (O2) -N(R' ) 2, -C(0)-OR', -N-S (O2 ) (R' ) , -NS02R^, -
C(0)N(R' ) (R^) , -NC(0)R^ -N(R')(R^), -C(0)R^, -
25 C{0)N=C(NH)2 or R^ .
Each R is independently selected from 5-8
membered aromatic or non-aromatic carbocyclic or
heterocyclic ring systems each optionally substituted
with R' , -C(0)R' or -C(0)OR'; or an 8-10 membered
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bicyclic ring system comprising aromatic carbocyclic
rings, aromatic heterocyclic rings or a combination of an
aromatic carbocyclic ring and an aromatic heterocyclic
ring each optionally substituted with R' , -C(0)R' or
5 C (O) OR^ .
R is selected from halogen, or a {C1-C3)
straight chain or branched alkyl.
Each Y is independently selected from N or C.
If either Y is N, then R or U attached to Y is a lone
10 pair of electrons.
Z is CH, N, C(0CH3), C (CH3) , CCNHs), C (OH) or
C(F) .
Each U is independently selected from R or J.
Each J is independently selected from a (C1-C4)
15 straight chain or branched alkyl derivative substituted
with T.
Each T is independently selected from either
0(V) or N(H) (V) .
Each V is independently selected from
20 C(0)N=C(R) (N(R)2)a wherein the two geminal R on the
nitrogen are optionally bound to one another to form a 4-
8 membered carbocyclic or heterocyclic ring.
When the two R components form a ring, it will
obvious to those skilled in the art that a terminal
25 hydrogen from each unfused R component will be lost. For
example, if a ring structure is .formed by binding those
two R components together, one being -CH3 and the other
being -CH2-CH3, one terminal hydrogen on each R component
(indicated in bold) will be lost. Therefore, the
30 resulting portion of the ring structure will have the
formula -CH2-CH2-CH2- .
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Each K is independently selected from a (C1-C4)
straight chain or branched alkyl derivative substituted
with D, or -0P(0) (OH) 2.
Each D is independently selected from either
5 enantiomer of
Each M is independently selected from either O
or NH.
Each G is independently selected from NH2, OH^
or H .
10 Each Re is independently selected from H, OH,
C(0)OH, (C1-C7) -straight or branched alkyl optionally
substituted with N(R')2, OR', CO2RS C0N(R')2^ or
5
S02N(R )2f or a 5-6 membered carbocyclic^ heterocyclic or
heteroaryl ring system optionally substituted with
15 N(RM2/ ORS CO2R' , C0N(R')2/ or S02N(R^)2. When G forms a
ring with Rs, it will be obvious to those skilled in the
art that a terminal hydrogen from the unfused G and Rg
component will be lost. For example, if a ring structure
is formed by binding the G and Re components together,
2 0 one being -NH2 and the other being -CH2-CH2-CH2-CH3, one
terminal hydrogen on each R component (indicated in bold)
will be lost. Therefore, the resulting portion of the
ring structure will have the formula -NH-CH2-CH2-CH2-CH2- .
In another embodiment, the invention provides
25 pharmaceutical compositions comprising the p3 8 and/or
ZAP70 inhibitors of this invention. These compositions
may be utilized in methods for treating or preventing a
variety of p38-mediated disorders, such as cancer,
inflammatory diseases, autoimmune diseases, destructive
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bone disorders, proliferative disorders, infectious
diseases, viral diseases and neurodegenerative diseases
or ZAP7 0-mediated disorders, including transplantation,
autoimune disease, cancer, multiple sclerosis, graft
5 versus host disease, and Kawasaki syndrome. These
compositions are also useful in methods for preventing
cell death and hyperplasia and therefore may be used to
treat or prevent reperf usion/ischemia in stroke, heart
attacks, and organ hypoxia. The compositions are also
10 useful in methods for preventing thrombin-induced
platelet aggregation. Each of these above-described
methods is also part of the present invention .
DETAILED DESCRIPTION OF THE INVENTION
15
These compounds have the general formula
(la) ,
(lb) ,
(Ic)
and (Id) ,
2 0 wher ein each of Qi and Q2 are independently selected from
a phenyl or 5-6 membered aromatic heterocyclic ring
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system, or a 8-10 membered bicyclic ring system
comprising aromatic carbocyclic rings, aromatic
heterocyclic rings or a combination of an aromatic
carbocyclic ring and an aromatic heterocyclic ring.
5 The rings that make up Qi are substituted with
1 to 4 substituents, each of which is independently
selected from halo; C1-C3 alkyl optionally substituted
with -NR' 2a OR', CO2R' or CONR'2; O- (C1-C3) -alkyl
optionally substituted with NR'2, OR', CO2R' or CONR'2;
10 NR'2; OCF3; CF3; NO2; CO2R' ; CONR' ; SR' ; S(02)N(R')2; SCF3;
CN; N(R')C(0)R*; N(R')C(0)OR^ N (R' ) C (O) C (O) R' ;
N(R' ) S (02)R*; N(R')R'; N(R')2; or'; OC(0)rS- OP(0)3H2; or
N=C-N(R' ) 2.
The rings that make up Q2 are optionally
15 substituted with up to 4 substituents, each of which is
independently selected from halogen; C1-C3 straight or
branched alkyl optionally substituted with R', NR'2/ OR',
CO2R' / S{02)N(R')2, N=C-N(R')2/ R^ 0-P(03)H2, or CONR'2; O-
(C1-C3) -alkyl; O- (C1-C3) -alkyl optionally substituted with
20 NR'2/ OR', CO2R'/ S(02)N(R')2/ N=CR ' -N (R' ) 2, R^ 0P(O3)H2,
or CONR' 2; NR'2; OCF3; CF3; NO2; CO2R' ; CONR'2; R^; OR^;
Nr\; SR^; C(0)R^• C(0)N(R')R^; C(0)OR^• SR' ; S{02)N{R')2;
SCF3; N=CR'-N{R' )2; 0R\- O-CO2R'; N(R')C(0)r\-
N(R' )C(0)0R'; N(R' )C(0)C(0)R'; N (R' ) S (O2) R' ; N(R')R^•
25 N(R^)2; ORS- OC(0)rS- 0P(0)3H2; K; or CN.
Each R' is independently selected from
hydrogen; (C1-C3) -alkyl; (C2-C3) -alkenyl or alkynyl;
phenyl or phenyl substituted with 1 to 3 substituents
independently selected from halo, methoxy, cyano, nitro,
30 amino, hydroxy, methyl or ethyl; or a 5-6 membered
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heterocyclic ring system optionally substituted with 1 to
3 substituents independently selected from halo, methoxy,
cyano, nitro, amino, hydroxy, methyl or ethyl.
Each R is independently selected from hydrogen,
5 -N(R^)2/ -0R^ SR^, -C{0)-N(r2)2. "S (O2) "N (R^) 2,
-C(0)-OR^ or -C(0)R^ wherein two adjacent R are optionally
bound to one another and, together with each Y to which
they are respectively bound, form a 4-8 membered
carbocyclic or heterocyclic ring.
10 Each R^ is independently selected from
hydrogen; or (C1-C3) -alkyl or (C1-C3) -alkenyl, each
optionally substituted with -N(RM2. -OR', SR' , -O-C(O)"
N(R')2/ -C(0)-N{R' )2/ -S (02)-N(R' )2, -C (O) -OR' , -NS02R\ -
NS02R^, -C(0)N(R') (R^) . -NC{0)r\ -N(R')(R^), -N(R')(R^)r -
15 C(0)R^, -C(0)N(R' ) (R^) , -N(R^)2/ -C(0)N=C(NH)2 or R .
Each R^ is independently selected from 5-8
membered aromatic or non-aromatic carbocyclic or
heterocyclic ring systems each optionally substituted
with R' , R^ -C(0)R', -C(0)r\ -C (O) OR^ or -K; or an 8-10
20 membered bicyclic ring system comprising aromatic
carbocyclic rings, aromatic heterocyclic rings or a
combination of an aromatic carbocyclic ring and an
aromatic heterocyclic ring each optionally substituted
with R^, R^, -C{0)R', -C(0)R^, -C{0)OR^ or -K.
25 Each R^ is independently selected from R' ; (Ci-
C7) -straight or branched alkyl optionally substituted
withR', N(R')2, OR', CO2R' / C0N(R')2, S02N(R')2or
S02N(R^)2; or a 5-6 membered carbocyclic or heterocyclic
ring system optionally substituted with N(R')2/ OR',
30 CO2R' f CON(R')2/ S02N(R')2or S02N(R^)2.
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5
Each R is independently selected from
hydrogen, (C1-C3) -alkyl, or (C1-C3) -alkenyl; each
optionally substituted with -N(R')2^ -OR', SR' , -C(0)-
N{R')2/ -S (O2) -N(R' )2, -C(0)-OR', -N-S (O2) (R' ) , -NSOsR^, -
5 C (O)N(R' ) (R^) , -NC(0)R', -N(R')(R^), -C(0)R^, -
C(0)N=C(NH)2 or R^ .
Each R is independently selected from 5-8
membered aromatic or non-aromatic carbocyclic or
heterocyclic ring systems each optionally substituted
10 with R' , -C(0)R' or -C(0)OR'; or an 8-10 membered
bicyclic ring system comprising aromatic carbocyclic
rings, aromatic heterocyclic rings or a combination of an
aromatic carbocyclic ring and an aromatic heterocyclic
ring each optionally substituted with R' , -C(0)R^ or
15 C(O)0R'.
7
R is selected from H, halogen, or a (C1-C3)
straight chain or branched alkyl.
Each Y is independently selected from N or C.
If either Y is N, then R or U attached to Y is a lone
20 pair of electrons.
Z is CH, N, C(OCH3), C (CH3) , C{NH2), C{OH) or
C(F) .
Each U is independently selected from R or J,
Each J is independently selected from a (C1-C4)
25 straight chain or branched alkyl derivative substituted
with T.
Each T is independently selected from either
0(V) or N(H) (V) ,
Each V is independently selected from
30 C(0)N=C(R) (N(R)2), wherein the two geminal R on the
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nitrogen are optionally bound to one another to form a 4-
8 membered carbocyclic or heterocyclic ring.
When the two R components form a ring, it will
obvious to those skilled in the art that a terminal
5 hydrogen from each unfused R component will be lost. For
example, if a ring structure is formed by binding those
two R components together, one being -CH3 and the other
being -CH2-CH3, one terminal hydrogen on each R component
(indicated in bold) will be lost. Therefore, the
10 resulting portion of the ring structure will have the
formula -^2-^2-0112-.
Each K is independently selected from a (Ca-C4)
straight chain or branched alkyl derivative substituted
with D, or -0P(0) (0H)2-
15 Each D is independently selected from either
enantiomer of
Each M is independently selected from either O
or NH.
Each G is independently selected from NH2/ OH,
20 or H.
Each Re is independently selected from H, OH,
C(0)OH, (C1-C7) -straight or branched alkyl optionally
substituted with N{R' ) 2. ORS CO2R' . C0N(R')2/ or
S02N{R^)2; or a 5-6 membered carbocyclic, heterocyclic or
25 heteroaryl ring system optionally substituted with
N(R')2/ OR', CO2RS C0N(R')2. or S02N(R^)2- When G forms a
ring with Rs. it will be obvious to those skilled in the
art that a terminal hydrogen from the unfused G and Rq
component will be lost. For example, if a ring structure
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is formed by binding the G and Rs components together,
one being -NH2 and the other being -CH2-CH2-CH2-CH3, one
terminal hydrogen on each R component (indicated in bold)
will be lost. Therefore, the resulting portion of the
5 ring structure will have the formula -NH-CH2-CH2-CH2-CH2- •
A heterocyclic ring system or a heterocyclic
ring contains 1 to 4 heteroatoms, which are independently
selected from O, and S. A substitutable nitrogen on
an aromatic or non-aromatic heterocyclic ring may be
10 optionally substituted. N or S may also exist in
oxidized form such as NO, SO and SO2 -
One having ordinary skill in the art will
recognize that the maximum number of heteroatoms in a
stable, chemically feasible heterocyclic ring, whether it
15 is aromatic or non-aromatic, is determined by the size of
the ring, degree of unsaturation, and valence of the
heteroatoms. In general, a heterocyclic ring may have
one to four heteroatoms so long as the heterocyclic ring
is chemically feasible and stable.
2 0 The term ''chemically stable arrangement" or
''chemically feasible and stable'' as used herein, refers
to a compound structure that renders the compound
sufficiently stable to allow manufacture and
administration to a mammal by methods known in the art,
25 Typically, such compounds are stable at a temperature of
40°C or less, in the absence of moisture or other
chemically reactive conditions, for at least a week.
According to a preferred embodiment, Qi
is selected from phenyl or pyridyl containing 1 to 3
30 substituents, wherein at least one of said substituents
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is in the ortho position and said substituents are
independently selected from chloro, fluoro^^ bromo;. -CH3,
-OCH3, -OH, -CF3, -OCF3, -0(CH2)2CH3/ NH2, 3,4-
methylenedioxy, -NCCHs) 2, -NH-S (O) 2-phenyl, ~NH-C (O) O-CH2-
5 4-pyridine, -NH-C (O) CHa-morpholine, -NH-C (O) CH2-N (CH3) z,
-NH-C (O) CH2-piperazine, -NH-C (O) CHg-pyrrolidine,
-NH-C (O) C (O) -morpholine, -NH-C (O) C (O) -piperazine,
-NH-C (O) C (O) -pyrrolidine, -0-C (O) CH2-N(CH3) 2/ or
-0-(CH2)2-N(CH3)2.
10 Even more preferred are phenyl or pyridyl
containing at least 2 of the above-indicated substituents
both being in the ortho position.
Some specific examples of preferred Qi are:
15
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NH2
.NH2
OCH3
■CI
NH2
OH
CI
CI
■OCH3
NH2
NH2
OH
.NH2
HaC^-r^CHa HaC^V^CHa HaC^-T^CH
00 Ha
•OH
HaCV^CHa HaCO'V^CHa CTV^OCH
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or
5 Most preferably, Qi is selected from 2-fluoro-
6-trif luoromethylphenyl, 2, 6-dif luorophenyl, 2, 6-
dichlorophenyl , 2-chloro-4-hydroxyphenyl , 2-chioro-4-
arainophenyl , 2, 6-dichloro-4-am.inophenyl, 2, 6-dichloro-3-
aminophenyl, 2, 6-dimethyl-4-hydroxyphenyl, 2-methoxy"-3, 5-
10 dichloro-4-pyridyl, 2-chloro-4,5 methylenedioxy phenyl,
or 2-chloro-4~ (N-2-morpholino-acetamido) phenyl .
According to a preferred embodiment, Q2 is
phenyl, pyridyl or naphthyl containing 0 to 3
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PCT/USOl/25015
substituents, wherein each substituent is independently
selected from chloro, fluoro, bromo, methyl, ethyl,
isopropyl, -OCH3, -OH, -NH2, -CF3, -OCF3, -SCH3, -OCH3,
-C(0)OH, -C(0)0CH3, -CH2NH2, -N(CH3)2, -CHz-pyrrolidine and
5 -CH2OH.
Some specific examples of preferred Q2 are:
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5
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PCT/USOl/25015
HNj^H
NH2
H2N
H2
NH "-NH
NH
O2H
O2H
CI
OH
10
unsubstituted 2-pyridyl or unsubstituted phenyl .
Most preferred are compounds wherein Q2 is
selected from phenyl, 2-isopropylphenyl, 3,4-
dimethylphenyl, 2-ethylphenyl, 3-f luorophenyl , 2-
methylphenyl, 3-chloro-4-f luorophenyl, 3-chlorophenyl,
carbomethoxylphenyl, 2-carboxyphenyl, 2-methyl-4-
2-
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chlorophenyl;. 2-broinophenyl;. 2-pyridyl, 2-
methylenehydroxyphenyl, 4-f luorophenyl, 2-methyl-4-
fluorophenyl, 2-chloro-4-f luorphenyl, 2, 4-dif luorophenyl,
2 -hydroxy- 4 - f luorphenyl ^ 2 -me thy 1 enehydr oxy- 4 -
5 fluorophenyl, 1-naphthyl, 3-chloro-2-methylenehydroxy, 3-
chloro-2-iaethyl, or 4-f luoro-2-inethyl .
According to another preferred embodiment, R*^
is a halogen. In a more preferred embodiment, r'^ is CI.
According to another preferred embodiment, each
10 Y is C.
According an even more preferred embodiment,
each Y is C and the R and U attached to each Y component
is hydrogen.
Some specific examples of preferred J are:
, and
According to another preferred embodiment, K is
a 0-4 atom chain terminating in an ester.
20 According to another preferred embodiment, M is
O.
Some specific examples of preferred K are:
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5 More preferably, K is selected from:
O O
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Some preferred embodiments are provided in
Tables 1 to 3 below:
Table 1
Cmpd
Nmbr
Structure
Cmpd
Nitibr
Structure
101
102
F
NH2 N
ir
o
I
N
103
104
H2N NH2
T
o
F I
NH2 N
T
O
r
N
NH.
105
106
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Table 1 ( cont . )
Cmpd
Niabr
Structure
Cmpd
Nrabr
Structure
107
F
M N
f
F
109
F [ F
NH2 N
108
Y
O
r
N
N
r
o
N
110
F y "F
.N N
f
F
N
N
Y
O
r
N
N
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Table 2 .
Cmpd
Nitibr
111
Structure
113
O
F T F
O^N
NH2 N
o
Cmpd
Nmbr
112
Structure
F
NH2 N
O
O^NH
T
o
114
F I
NH2 N
HO
O
O^NH
T
O
115
117
F
NH2 N
HO^^O
116
Y
o
NH
F J F
NH2 N
-O^NH
T
o
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Table 3.
Cmpd
Nitibr
Structure
118
Cmpd
Nmbr
119
NH2
CI
Structure
NH2
NH.
0^0
120
122
124
©Y^
NH2
OH
k./NH2
O
CI
oh
NH2
0^0
CI
NH2
CI
121
123
125
F 1^
NH2
"../NH2
o ^O
CI
^Y^
NH2
OH
I^OH
^-o
CI
NH2 N
HOaa, NH
■0^0
CI
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Particularly preferred embodiments include:
5
10 Particularly preferred embodiments also
include :
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Other particnlarly preferred embodiments
include :
Other particularly preferred embodiments
include :
O
NH2
10 wherein X
or
O
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Other particularly preferred embodiments
include
O
NH2
wherein X =
or
O
HO-P
ho""
Other particularly preferred embodiments
10 include:
N
/
wherein X is N(CH3)2/
>3
N NWle
or
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Other particularly preferred embodiments
include :
5 wherein Y = Me or H; and X = (CH2)3/ CH2C (CH3) 2CH2,
CH2N (Me) C (O) CH2.
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According to another embodiment, the present
invention provides methods of producing the above-
identified compounds of the formulae (la), (lb), (Ic) or
(Id) . Representative synthesis schemes are depicted
5 below. In all schemes, the LI and L2 groups on the
initial materials are meant to represent leaving groups
ortho to the nitrogen atom in a heterocyclic ring. For
example, compound A may be 2, 6-dichloro-3 nitro pyridine.
Scheme 1
10
One having skill in the art will recognize
Scheme 1 may be used to synthesize compounds having the
15 general formula of (la), (lb), (Ic) and (Id).
According to another embodiment of the
invention, the activity of the p38 inhibitors of this
invention may be assayed in vitro ^ in vivo or in a cell
20 line. In vitro assays include assays that determine
inhibition of either the kinase activity or ATPase
activity of activated p38. Alternate in vitro assays
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quantitate the ability of the inhibitor to bind to p38
and may be measured either by radiolabelling the
inhibitor prior to binding, isolating the inhibitor/p38
complex and determining the amount of radiolabel bound,
5 or by running a competition experiment where new
inhibitors are incubated with p38 bound to known
radioligands .
Cell culture assays of the inhibitory effect of
the compounds of this invention may determine the amounts
10 of TNF, IL-1, IL-6 or IL-8 produced in whole blood or
cell fractions thereof in cells treated with inhibitor as
compared to cells treated with negative controls. Level
of these cytokines may be determined through the use of
commercially available ELISAs.
15 An in vivo assay useful for determining the
inhibitory activity of the p38 inhibitors of this
invention are the suppression of hind paw edema in rats
with Mycobacterium butyrl cum- Indiuced adjuvant arthritis.
This is described in J.C. Boehm et al . , J. Med. Chem. ,
20 39, pp. 3929-37 (1996), the disclosure of which is herein
incorporated by reference. The p38 inhibitors of this
invention may also be assayed in animal models of
arthritis, bone resorption, endotoxin shock and immune
function, as described in A. M. Badger et al . , J.
25 Pharmacol. Experimental Therapeutics, 279, pp. 1453-61
(1996) , the disclosure of which is herein incorporated by
reference .
The p38 inhibitors or pharmaceutical salts
thereof may be formulated into pharmaceutical
30 compositions for administration to animals or humans.
These pharmaceutical compositions, which comprise an
amount of p38 inhibitor effective to treat or prevent a
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p38-mediated condition and a pharmaceutically acceptable
carrier, are another embodiment of the present invention.
The term ^^pSS-mediated condition", as used
herein means any disease or other deleterious condition
5 in which p38 is known to play a role. This includes
conditions known to be caused by IL-1, TNF, IL-6 or IL-8
overproduction. Such conditions include, without
limitation, inflammatory diseases, autoimmune diseases,
destructive bone disorders, proliferative disorders,
10 infectious diseases, neurodegenerative diseases,
allergies, reperfusion/ischemia in stroke, heart attacks,
angiogenic disorders, organ hypoxia, vascular
hyperplasia, cardiac hypertrophy, thrombin-induced
platelet aggregation, and conditions associated with
15 prostaglandin endoperoxidase synthase-2.
Inflammatory diseases which may be treated or
prevented by the compounds of this invention include, but
are not limited to, acute pancreatitis, chronic
pancreatitis, asthma, allergies, and adult respiratory
2 0 distress syndrome.
Autoimmune diseases which may be treated or
prevented by the compounds of this invention include, but
are not limited to, glomerulonephritis, rheumatoid
arthritis, systemic lupus erythematosus, scleroderma,
25 chronic thyroiditis. Graves' disease, autoimmune
gastritis, diabetes, autoimmune hemolytic anemia,
autoimmune neutropenia, thrombocytopenia, atopic
dermatitis, chronic active hepatitis, myasthenia gravis,
multiple sclerosis, inflammatory bowel disease,
30 ulcerative colitis, Crohn^ s disease, psoriasis, or graft
vs. host disease.
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Destructive bone disorders which may be treated
or prevented by the compounds of this invention include,
but are not limited to, osteoporosis, osteoarthritis and
multiple myeloma-related bone disorder.
5 Proliferative diseases which may be treated or
prevented by the compounds of this invention include, but
are not limited to, acute myelogenous leukemia, chronic
myelogenous leukemia, metastatic melanoma, Kaposi's
sarcoma, and multiple myeloma.
10 Angiogenic disorders which may be treated or
prevented by the compounds of this invention include
solid tumors, ocular neovasculization, infantile
haemangiomas .
Infectious diseases which may be treated or
15 prevented by the compounds of this invention include, but
are not limited to, sepsis, septic shock, and
Shigellosis .
Viral diseases which may be treated or
prevented by the compounds of this invention include, but
20 are not limited to, acute hepatitis infection (including
hepatitis A, hepatitis B and hepatitis C) , HIV infection
and CMV retinitis.
Neurodegenerative diseases which may be treated
or prevented by the compounds of this invention include,
25 but are not limited to, Alzheimer's disease, Parkinson's
disease, cerebral ischemias or neurodegenerative disease
caused by traumatic injury.
^^p38-mediated conditions" also include
ischemia/reperfusion in stroke, heart attacks, myocardial
30 ischemia, organ hypoxia, vascular hyperplasia, cardiac
hypertrophy, and thrombin- induced platelet aggregation.
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In addition, p38 inhibitors of the instant
invention are also capable of inhibiting the expression
of inducible pro-inflammatory proteins such as
prostaglandin endoperoxide sYnthase-2 (PGHS-2), also
5 referred to as cyclooxygenase-2 (COX-2) . Therefore, other
^^p38-mediated conditions" which may be treated by the
compounds of this invention include edema, analgesia,
fever and pain, such as neuromuscular pain, headache,
cancer pain, dental pain and arthritis pain.
10 The diseases that may be treated or prevented
by the p38 inhibitors of this invention may also be
conveniently grouped by the cytokine (IL~1, TNF, IL-6,
IL-8) that is believed to be responsible for the disease.
Thus, an IL-l-mediated disease or condition
15 includes rheumatoid arthritis, osteoarthritis, stroke,
endotoxemia and/or toxic shock syndrome, inflammatory
reaction induced by endotoxin, inflammatory bowel
disease, tuberculosis, atherosclerosis, muscle
degeneration, cachexia, psoriatic arthritis, Reiter's
20 syndrome, gout, traumatic arthritis, rubella arthritis,
acute synovitis, diabetes, pancreatic B-cell disease and
Alzheimer' s disease .
TNF-mediated disease or condition includes,
rhetimatoid arthritis, rheumatoid spondylitis,
25 osteoarthritis, gouty arthritis and other arthritic
conditions, sepsis, septic shock, endotoxic shock, gram
negative sepsis, toxic shock syndrome, adult respiratory
distress syndrome, cerebral malaria, chronic pulmonary
inflammatory disease, silicosis, pulmonary sarcoidosis,
30 bone resorption diseases, reperfusion injury, graft vs.
host reaction, allograft rejections, fever and myalgias
due to infection, cachexia secondary to infection, AIDS,
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ARC or malignancy, keloid formation, scar tissue
formation, Crohn's disease, ulcerative colitis or
pyresis. TNF-mediated diseases also include viral
infections, such as HIV, CMV, influenza and herpes; and
5 veterinary viral infections, such as lentivirus
infections, including, but not limited to equine
infectious anemia virus, caprine arthritis virus, visna
virus or maedi virus; or retrovirus infections, including
feline immunodeficiency virus, bovine immunodeficiency
10 virus, or canine immunodeficiency virus,
IL-8 mediated disease or condition includes
diseases characterized by massive neutrophil
infiltration, such as psoriasis, inflammatory bowel
disease, asthma, cardiac and renal reperfusion injury,
15 adult respiratory distress syndrome, thrombosis and
glomerulonephritis .
In addition, the compounds of this invention
may be used topically to treat or prevent conditions
caused or exacerbated by IL-1 or TNF. Such conditions
20 include inflamed joints, eczema, psoriasis, inflammatory
skin conditions such as sunburn, inflammatory eye
conditions such as conjunctivitis, pyresis, pain and
other conditions associated with inflammation.
According to another embodiment, the compounds
25 of this invention may be used to treat ZAP7 0-mediated
conditions including, without limitation, organ or tissue
rejection associated with transplantation, autoimune
disease, e.g., rheumatoid arthritis, systemic lupus
erythematosus (SLE) , psoriasis, Sjogren^s Syndrome,
30 thyroiditis, pulmonary fibrosis, bronchiolitis
obliterans, hemolytic anemia and Wegener ^s
granulomatosis, cancer, including leukemia and lymphoma.
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multiple sclerosis, graft versus host disease, and
Kawasaki syndrome .
The ZAP7 0 inhibitors or pharmaceutical salts
thereof may be formulated into pharmaceutical
5 compositions for administration to animals or humans.
These pharmaceutical compositions, which comprise an
amount of ZAP70 inhibitor effective to treat or prevent a
ZAP7 0-mediated condition and a pharmaceutically
acceptable carrier, are another embodiment of the present
10 invention.
In addition to the compounds of this invention,
pharmaceutically acceptable salts of the compounds of
this invention may also be employed in compositions to
treat or prevent the above-identified disorders.
15 Pharmaceutically acceptable salts of the
compounds of this invention include those derived from
pharmaceutically acceptable inorganic and organic acids
and bases. Examples of suitable acid salts include
acetate, adipate, alginate, aspartate, benzoate,
20 benzenesulfonate, bisulfate, butyrate, citrate,
camphorate, camphorsulf onate, cyclopentanepropionate,
digluconate, dodecylsulf ate, ethanesulf onate, formate,
fumarate, glucoheptanoate, glycerophosphate, glycolate,
hemisulfate, heptanoate, hexanoate, hydrochloride,
2 5 hydrobromide , hydroiodide , 2-hydroxyethane sul f onate ,
lactate, maleate, malonate, methanesulf onate, 2-
naphthalenesulf onate, nicotinate, nitrate, oxalate,
palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate,
30 succinate, sulfate, tartrate, thiocyanate, tosylate and
undecanoate. Other acids, such as oxalic, while not in
themselves pharmaceutically acceptable, may be employed
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in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their
pharmaceutically acceptable acid addition salts.
Salts derived from appropriate bases include alkali metal
5 (e.g., sodium and potassi\im) , alkaline earth metal (e.g.,
magnesium), ammonium and N~(Cl-4 alkyl)4+ salts. This
invention also envisions the quaternization of any basic
nitrogen-containing groups of the compounds disclosed
herein. Water or oil-soluble or dispersible products may
10 be obtained by such quaternization.
Pharmaceutically acceptable carriers that may
be used in these pharmaceutical compositions include, but
are not limited to, ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum
15 albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride
mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium
hydrogen phosphate, potassium hydrogen phosphate, sodium
20 chloride, zinc salts, colloidal silica, magnesium
tri silicate, polyvinyl pyrrol idone, cellulose-based
substances, polyethylene glycol, sodium
carboxymethyl cellulose, polyacrylates , waxes,
polyethylene-polyoxypropylene-block polymers ,
25 polyethylene glycol and wool fat.
The compositions of the present invention may
be administered orally, parenterally, by inhalation
spray, topically, rectally, nasally, buccally, vaginally
or via an implanted reservoir. The term "parenteral" as
3 0 used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-synovial,
intr asternal, intrathecal, intrahepatic, intralesional
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and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously.
Sterile injectable forms of the compositions of
5 this invention may be aqueous or oleaginous suspension.
These suspensions may be formulated according to
techniques known in the art using suitable dispersing or
wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable
10 solution or suspension in a non-toxic parenterally-
acceptable diluent or solvent, for example as a solution
in 1, 3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water. Ringer's
solution and isotonic sodiiim chloride solution. In
15 addition, sterile, fixed oils are conventionally employed
as a solvent or suspending medium. For this purpose, any
bland fixed oil may be employed including synthetic mono-
or di-glycerides . Fatty acids, such as oleic acid and
its glyceride derivatives are useful in the preparation
2 0 of injectables, as are natural pharmaceutically-
acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil
solutions or suspensions may also contain a long-chain
alcohol diluent or dispersant, such as carboxymethyl
25 cellulose or similar dispersing agents which are commonly
used in the formulation of pharmaceutically acceptable
dosage forms including emulsions and suspensions. Other
commonly used surfactants, such as Tweens, Spans and
other emulsifying agents or bioavailability enhancers
3 0 which are commonly used in the manufacture of
pharmaceutically acceptable solid, liquid, or other
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dosage forms may also be used for the purposes of
formulation.
The pharmaceutical compositions of this
invention may be orally administered in any orally
5 acceptable dosage form including, but not limited to,
capsules, tablets, aqueous suspensions or solutions- In
the case of tablets for oral use, carriers commonly used
include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added.
10 For oral acSministration in a capsule form, useful
diluents include lactose and dried cornstarch. When
aqueous suspensions are required for oral use, the active
ingredient is combined with emulsifying and suspending
agents. If desired, certain sweetening, flavoring or
15 coloring agents may also be added.
Alternatively, the pharmaceutical compositions
of this invention may be administered in the form of
suppositories for rectal administration. These can be
prepared by mixing the agent with a suitable non-
20 irritating excipient which is solid at room temperature
but liquid at rectal temperature and therefore will melt
in the rectum to release the drug. Such materials
include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this
25 invention may also be administered topically, especially
when the target of treatment includes areas or organs
readily accessible by topical application, including
diseases of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily
30 prepared for each of these areas or organs .
Topical application for the lower intestinal
tract can be effected in a rectal suppository formulation
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(see above) or in a suitable enema formulation.
Topically-transdermal patches may also be used.
For topical applications, the pharmaceutical
compositions may be formulated in a suitable ointment
5 containing the active component suspended or dissolved in
one or more carriers. Carriers for topical
administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol,
10 polyoxyethylene, polyoxypropylene compound, emulsifying
wax and water. Alternatively, the pharmaceutical
compositions can be formulated in a suitable lotion or
cream containing the active components suspended or
dissolved in one or more pharmaceutically acceptable
15 carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60,
cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,
benzyl alcohol and water.
For ophthalmic use, the pharmaceutical
20 compositions may be formulated as micronized suspensions
in isotonic, pH adjusted sterile saline, or, preferably,
as solutions in isotonic, pH adjusted sterile saline,
either with or without a preservative such as
benzylalkonium chloride. Alternatively, for ophthalmic
25 uses; the pharmaceutical compositions may be formulated
in an ointment such as petrolatum.
The pharmaceutical compositions of this
invention may also be administered by nasal aerosol or
inhalation. Such compositions are prepared according to
30 techniques well-known in the art of pharmaceutical
formulation and may be prepared as solutions in saline,
employing benzyl alcohol or other suitable preservatives.
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absorption promoters to enhance bioavailability,
f luorocarbons, and/or other conventional solubilizing or
dispersing agents .
The amount of p38 or ZAP70 inhibitor that may
5 be combined with the carrier materials to produce a
single dosage form will vary depending upon the host
treated and the particular mode of administration.
Preferably, the compositions should be formulated so that
a dosage of between 0.01 - 100 mg/kg body weight/day of
10 the inhibitor can be administered to a patient receiving
these compositions .
It should also be understood that a specific
dosage and treatment regimen for any particular patient
will depend upon a variety of factors, including the
15 activity of the specific compound employed, the age, body
weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and
the judgment of the treating physician and the severity
of the particular disease being treated. The amount of
2 0 inhibitor will also depend upon the particular compound
in the composition.
According to another embodiment, the invention
provides methods for treating or preventing a p38-
mediated condition comprising the step of administering
25 to a patient one of the above-described pharmaceutical
compositions. The term ^""patienf , as used herein, means
an animal, preferably a human.
Preferably, that method is used to treat or
prevent a condition selected from inflammatory diseases,
30 autoimmune diseases, destructive bone disorders,
proliferative disorders, infectious diseases,
degenerative diseases, allergies, reperf usion/ischemia in
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stroke, heart attacks, angiogenic disorders, organ
hypoxia, vascular hyperplasia, cardiac hypertrophy, and
thrombin-induced platelet aggregation.
According to another embodiment, the inhibitors
5 of this invention are used to treat or prevent an IL-1,
IL-6, IL-8 or TNF-mediated disease or condition. Such
conditions are described above.
Depending upon the particular p3 8-inediated
condition to be treated or prevented, additional drugs,
10 which are normally administered to treat or prevent that
condition, may be administered together with the
inhibitors of this invention. For example,
chemotherapeutic agents or other anti-prolif erative
agents may be combined with the p38 inhibitors of this
15 invention to treat proliferative diseases.
Those additional agents may be administered
separately, as part of a multiple dosage regimen, from
the p38 inhibitor-containing composition. Alternatively,
those agents may be part of a single dosage form, mixed
20 together with the p38 inhibitor in a single composition.
According to another embodiment, the invention
provides methods for treating or preventing a ZAP7 0-
mediated condition comprising the step of administering
to a patient one of the above-described pharmaceutical
25 compositions.
In order that the invention described herein
may be more fully understood, the following examples are
set forth. It should be understood that these examples
are for illustrative purposes only and are not. to be
30 construed as limiting this invention in any manner.
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EXAMPLE 1
Synthesis of p38 Inhibitor Compound 7
CHO
1
5
To a solution of LDA (60itimol, 40inLs) at -78° C,
was added dropwise a solution of 2 , 6-dibromopyridine
(40iniriol, 9.48gms) in THE (30mLs, dried). The mixture was
stirred at -78° C for 20 minutes. Ethyl formate (400mmol,
10 32.3mLs) was added and stirring was continued at -78° C
for 2 hours. Saturated ammonium chloride (200mLs) was
added and the mixture was warmed to room temperature .
The reaction mixture was diluted with ethyl acetate and
the organic layer was washed with aqueous acid and base.
15 The organic layer was dried and evaporated in vacuo. The
resulting material was purified by flash chromatography
on silica gel followed by eluting with 10% ethyl acetate
in n-hexane to afford 1 (32mmol, 8.41gms) as a white
solid.
20 12
A solution of 1 (77 6 mmol, 2 05.6 gms) and
triethyl orthoformate (200 mL) dissolved in ethanol (750
mL) was refluxed overnight. The reaction mixture was
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cooled, and evaporated in vacuo. The remaining red oil
was dissolved in hexane and filtered over a plug of
silica gel. The plug was eluted with 50% CHsCls/hexane .
The filtrate was evaporated to afford 2 as an oil.
5 2 3
To a suspension of 60% NaH (130 imnol, 5.20 g)
and 2 (61.2 mmol, 20.76 g) in THF (100 mL) at reflux was
added dropwise a solution of 2 , 6-dif luoroaniline (61.3
10 mmol, 20 g) in THF (100 mL) . After the aniline had been
added, Pd(PPh3)4 (100 mg) was added. The mixture was
refluxed for one hour and cooled. Hydrochloric acid (IN,
100 mL) was added and stirring was continued for one
hour. The reaction mixture was extracted with CH2CI2.
15 The organic extract was dried and evaporated In vacuo.
The resulting material was dissolved in a minimal amount
of CH2CI2 and hexane was added. The solution was cooled
precipitating 3 as a yellow solid.
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p-f luorophenylboronic acid (57.5 mmol, 8.05 g) ,
and 3 (45.9 mmol, 14,70 g) were dissolved in a
5 dimethoxyethane (300 mL) . Cesium fluoride (68.6 mmol/
10.42 g) and tetrakis (triphenylphosphine) palladium (0)
(100 mgs) were added to the solution and the suspension
was allowed to reflux overnight. The reaction mixture
was poured into water and extracted with CH2CI2- The
10 organic extract was washed with IN NaOH^ dried with MgSOo
and filtered over a plug of silica gel. The plug was
eluted with CH2CI2 and the filtrate was evaporated in
vacuo. The resulting yellow solid was triturated with
50% CH2Cl2/hexane to afford 4 (9.50g, 62%) as a yellow
15 solid.
20
A solution of 4 (70.1 mmol, 23.01 g) in toluene
(250 mL) was combined with a 20% solution of phosgene in
toluene (151 mmol, 80 mL) and heated to reflux for two
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hours. The reaction was cooled and poured into ammonium
hydroxide. The mixture was stirred for five minutes and
extracted with methylene chloride. The organic extract
was dried and filtered over a plug of silica gel. The
5 plug was eluted with methylene chloride to remove
residual starting material. It was then eluted with 50%
ethyl acetate/methylene chloride to obtain 5. The
filtrate was evaporated in vacuo to afford 5 (21.38 g,
86%) as a white solid.
10 5 6
Sodium borohydride (36.5 mmol, 1.38 g) was
added to a solution of 5 (60.0 mmol, 21.38 g) in THF (100
mL) and the solution was stirred for one hour at 0°C and
th en two hours at room temperature . The reaction was
15 poured into IN HCl and extracted with methylene chloride.
The organic extract was dried and filtered over a plug of
silica gel. The plug was eluted with 5% ethyl
acetate/methylene chloride to remove residual starting
material. It was then eluted with ethyl acetate to
20 obtain 6, The filtrate was evaporated to afford 6 as a
white solid.
The spectral data for compound 6 was :
NMR (500 MHz, CDCI3) 5 7.90 (d, IH) , 7.60 (d, 2H) , 7.5-
7.3 (m, 5H) , 6.30 (d, 2H) , 4.5 (s, 2H) , 2.3 (s, 2H) .
25
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A solution of 6 (2.79 mmol, 1.00 g) and p-
nitrophenyl chlorof ormate (5.56 itimol, 1.12 g) was cooled
5 to 0°C. Triethylamine (14.3 mmol, 2.0 mL) was added and
the solution was stirred for 15 minutes and poured into
ammonium hydroxide. The solution mixture was poured into
water and extracted with methylene chloride. The organic
extract was washed with saturated aqueous sodium
10 bicarbonate, dried, and evaporated in vacuo to afford 7
(730 mg, 65%) as a white solid.
EXAMPLE 2
Synthesis of p38 Inhibitor Prodrugs 9 and 10
15
A mixture of 8 (1.0 g, 2.30 mmol) and N,N-
dimethylf ormamide dimethyl acetal (1.01 g, 6.91 mmol) in
20 10 mL of toluene was heated to 80°C for 20 minutes. The
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resulting solution was cooled to room temperature.
Normal workup followed by chromatography on silica gel
(hexane/EtOAc: 10/4) gave amidine 9 (compound 101 of
Table 1) as a white solid. The spectral data for
5 compound 9 was: NMR (500 MHz, CDCI3) 6 8.3 (s, IH) , 7.7
(d, IH), 7.5-7.4 (m, IH) , 7.1-7.0 (m, IH) , 6.95-6.85 (t,
2H) , 6.85-6.75 (m, IH) , 6.45-6.4 (d, IH) , 6.2 (s, IH) ,
4.95 (s, 2H) , 3.05 (s, 3H) , 2.95 (s, 3H) .
dimethyl formamide dimethyl acetal (3.3 g, 22.4 mmol) in
10 mL of toluene was heated to 80°C for 90 minutes. The
resulting solution was cooled to room temperature.
15 Normal workup followed by chromatography on silica gel
(hexane/EtOAc: 2/1) gave bis-amidine 10 (compound 107 of
Table 1) as a white solid. The spectral data for
compound 10 was: ^H NMR (500 MHz, CDCI3) 5 8.4 (s, IH) ,
8.3 (s, IH), 8.05-7.95 (s, IH) , 7-15-7.05 (m, 2H) , 6.85-
20 6.75 (t, 2H) , 6.75-6.65 (m, 4H) , 4.95 (s, 2H) , 3.0-2.95
(d, 9H) , 2.65 (s, 3H) .
10
8 10
A mixture of 8 (1.0 g, 2.30 mmol) and N,N-
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EXAMPLE 3
Synthesis of p38 Inhibitor Prodrug 13
6 11
5
To a mixture of 6 (1.2 5gia, 3.35mmol) and 4-
nitrophenyl chlorof oritiate (O.Slgm, 4.02inmol) in
tetrahydrofuran (30mL) was added triethylamine (1.16mL,
8.38inmol) dropwise at 0°C . The resulting slurry was
10 allowed to stir at 0°C for 30 minutes. Ethanolamine
(0.6mL, lO.Ommol) was added and the solution was stirred
at 0°C for 30 minutes. Normal work-up followed by
chromatography on silica (hexane/acetone : 10/4) gave 11
(1.03gm, 2.23mmol) as a white solid. NMR (500 MHz,
15 CDCI3) 7.75 (d, IH) , 7.65-7.55 (m, 2H) , 7.5-7.4 (m, IH) ,
7.25-7.15 (t, 2H), 7.15-7.05 (t, 2H) , 6.4 (d, IH) , 5.2-
5.1 (bs, IH) , 5.15 {s,2H), 3.75-3.65 (t, 2H) , 3.4-3.3 (m,
2H) .
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10
A mixture of 11 (1.03gm, 2 . 23itimol) , (L) -BOC-
Val-OH (0.97gm, 4.4 6inmol), and 1- (3-dimethylaminopropyl )
S-ethylcarbodiimide hydrochloride in methylene chloride
(30mL) was stirred at room temperature for 1.5 hours.
Normal work-up followed by chromatography on silica
(hexane/ acetone: 10/4) gave Val deriv. 12 (1.38gms,
2.09inmol) as a white solid. NMR (500 MHz, CDCI3) 7.75
(d, IH) , 7.55-7.55 (m, 2H) , 7.5-7.4 (m, IH) , 7.25-7.15
(t, 2H) , 7,15-7.05 (t, 2H) , 6.4 (d, IH) , 5.40-5.35 (bs,
IH) , 5.05 (s, 2H), 5.00-4.95 (d, IH) , 4.4-4.3 (m, IH) ,
4.25-4.15 (m, IH) , 4.15-4.05 (m, IH) , 3.55-3.45 (m, 2H) ,
2.15-2.05 (m, IH) , 1.45 (s, 9H) , 1.0-0.85 (m, 6H) .
15
20
HtjJ
12
13
25
To a solution of 12 (1.38gms, 2.09mmol) in
methylene chloride (2 0mLs) was added trif luoroacetic acid
(lOmLs) . The solution was allowed to stir at room
temperature for 1 hour. Normal work-up gave a white solid
that was converted to its hydrochloride salt to give 13
(compound 111 of Table 2; 0.61gms, 1.02mmol) as a white
solid. The spectral data for compound 13 was: H NMR
(500 MHz, CDCI3) 7,65 (d, IH) , 7.55-7.45 (m, 2H) , 7.4-7.3
(m, IH) , 7.15-7.05 (m, 2H) , 7.05-5.95 (m, 2H) , 6.35 (d,
IH), 5.05-5.00 (bs, IH), 4.95 (s, 2H) , 4.15-4.05 (m, 2H) ,
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3.45-3.25 (III, 2H) , 3.2 (s, IH) , 1.95-1.85 (m, IH) , 0.90-
0.75 (m, 6H) .
EXAMPLE 4
Cloning of p38 Kinase in Insect Cells
5 Two splice variants of human p38 kinase, CSBPl
and CSBP2, have been identified. Specific
oligonucleotide primers were used to amplify the coding
region of CSBP2 cDNA using a HeLa cell library
(Stratagene) as a template. The polymerase chain
10 reaction product was cloned into the pET-15b vector
(Novagen) . The baculovirus transfer vector, pVL-(His)6-
p3 8 was constructed by subcloning a Xjbal-BamHI fragment
of pET15b- (His) 6-p38 into the complementary sites in
plasmid pVL1392 (Pharmingen) .
15 The plasmid pVL- (His ) 6-p38 directed the
synthesis of a recombinant protein consisting of a 23-
residue peptide (MGSSHHHHHHSSGLVPRGSHMLE, where LVPRGS
represents a thrombin cleavage site) fused in frame to
the N- terminus of p3 8, as confirmed by DNA sequencing and
2 0 by N- terminal sequencing of the expressed protein.
Monolayer culture of Spodoptera fruglperda (Sf9) insect
cells (ATCC) was maintained in TNM-FH medium (Gibco BRL)
supplemented with 10% fetal bovine serum in a T-flask at
27°C. Sf9 cells in log phase were co-transf ected with
25 linear viral DNA of Autographa calif onica nuclear
polyhedrosis virus (Pharmingen) and transfer vector pVL-
(His) 6-p38 using Lipofectin (Invitrogen) . The individual
recombinant baculovirus clones were purified by plaque
assay using 1% low melting agarose.
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EXAMPLE 5
Expression and Purification of Recombinant p38 Kinase
Trichoplusia ni (Tn-3 68) High-Five^ cells
(Invitrogen) were grown in suspension in Excel-405
protein free medium (JRH Bioscience) in a shaker flask at
27''C. Cells at a density of 1.5 X 10^ cells/ml were
5 infected with the recombinant baculovirus described above
at a multiplicity of- infection of 5. The expression
level of recombinant p38 was monitored by immunoblotting
using a rabbit anti-p38 antibody (Santa Cruz
Biotechnology) . The cell mass was harvested 72 hours
10 after infection when the expression level of p38 reached
its maximum.
Frozen cell paste from cells expressing the
(His) g-tagged p38 was thawed in 5 volumes of Buffer A (50
mM NaH2P04 pH 8.0, 200 mM NaCl, 2mM B-Mercaptoethanol , 10%
15 Glycerol and 0 . 2 mM PMSF) . After mechanical disruption
of the cells in a microf luidizer , the lysate was
centrifuged at 30,000 x g for 30 minutes. The
supernatant was incubated batchwise for 3-5 hours at 4^C
with Talon™ (Clontech) metal affinity resin at a ratio of
20 1 ml of resin per 2-4 mgs of expected p38. The resin was
settled by centrifugation at 500 x g for 5 minutes and
gently washed batchwise with Buffer A. The resin was
slurried and poured into a column (approx. 2.6 x 5.0 cm)
and washed with Buffer A + 5 mM imidazole.
25 The (His)6-p38 was eluted with Buffer A + 100
mM imidazole and subsequently dialyzed overnight at 4^C
against 2 liters of Buffer B, (50 mM HEPES, pH 7.5, 25 mM
^-glycerophosphate, 5% glycerol, 2mM DTT) . The Hisg tag
was removed by addition of at 1.5 units thrombin
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(Calbiochem) per mg of p38 and incubation at 20°C for 2-3
hours. The thrombin was quenched by addition of 0,2 inM
PMSF and then the entire sample was loaded onto a 2 ml
benzamidine agarose (American International Chemical)
5 column.
The flow through fraction was directly loaded
onto a 2.6 X 5.0 cm Q-Sepharose (Pharmacia) column
previously equilibrated in Buffer B + 0 . 2 mM PMSF. The
p38 was eluted with a 20 column volume linear gradient to
10 0.6M NaCl in Buffer B. The eluted protein peak was
pooled and dialyzed overnight at vs. Buffer C (50 mM
HEPES pH 7.5, 5% glycerol, 50 mM NaCl, 2 mM DTT, 0 . 2 mM
PMSF) .
The dialyzed protein was concentrated in a
15 Centriprep (Amicon) to 3-4 ml and applied to a 2 . 6 x 100
cm Sephacryl S-IOOHR (Pharmacia) column. The protein was
eluted at a flow rate of 35 ml/hr. The main peak was
pooled, adjusted to 20 mM DTT, concentrated to 10-80
mgs/ml and frozen in aliquots at -70*^C or used
2 0 immediately.
EXAMPLE 6
Activation of p38
p38 was activated by combining 0.5 mg/ml p38
with 0.005 mg/ml DD-double mutant MKK6 in Buffer B + lOmM
25 MgCl2, 2mM ATP, 0 . 2mM Na2V04 for 30 minutes at 20''c. The
activation mixture was then loaded onto a 1.0 x 10 cm
MonoQ column (Pharmacia) and eluted with a linear 20
column volume gradient to 1 . 0 M NaCl in Buffer B. The
activated p38 eluted after the ADP and ATP, The
30 activated p38 peak was pooled and dialyzed against buffer
B + 0.2mM Na2V04 to remove the NaCl. The dialyzed protein
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was adjusted to 1 . IM potassium phosphate by addition of a
4 . OM stock solution and loaded onto a 1.0 x 10 cm HIC
(Rainin Hydropore) column previously equilibrated in
Buffer D (10% glycerol, 20raM B-glycerophosphate, 2 . OmM
5 DTT) + I.IMK2HPO4. The protein was eluted with a 20
column volume linear gradient to Buffer D + 50mM K2HPO4 .
The double phosphorylated p38 eluted as the main peak and
was pooled for dialysis against Buffer B + 0 . 2mM Na2V04 .
The activated p38 was stored at -70^C.
10
EXAMPLE 7
p38 Inhibition Assays
A. Inhibition of Phosphorylation of EGF Receptor
Peptide
15
This assay was carried out in the presence of
10 mM MgCl2, 25 mM ^-glycerophosphate, 10% glycerol and
100 mM HEPES buffer at pH 1.6. For a typical IC50
determination, a stock solution was prepared containing
2 0 all of the above components and activated p3 8 (5 nM) .
The stock solution was aliquotted into vials. A fixed
volume of DMSO or inhibitor in DMSO (final concentration
of DMSO in reaction was 5%) was introduced to each vial,
mixed and incubated for 15 minutes at room temperature •
25 EGF receptor peptide, KRELVEPLTPSGEAPNQALLR, a phosphoryl
acceptor in p38-catalyzed kinase reaction (1), was added
to each vial to a final concentration of 200 jiM, The
kinase reaction was initiated with ATP (100 toM) and the
vials were incubated at 3 0''c. After 3 0 minutes, the
30 reactions were quenched with equal volume of 10%
trifluoroacetic acid (TEA) .
The phosphorylated peptide was quantified by
HPLC analysis. Separation of phosphorylated peptide from
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the unphosphorylated peptide was achieved on a reverse
phase column (Deltapak, 5 lom, C18 lOOD, Part no. 011795)
with a binary gradient of water and acteonitrile, each
containing 0.1% TFA. IC50 (concentration of inhibitor
5 yielding 50% inhibition) was determined by plotting the
percent {%) activity remaining against inhibitor
concentration .
B. Inhibition of ATPase Activity
10 This assay is carried out in the presence of 10
mM MgClzf 25 mM B-glycerophosphate, 10% glycerol and 100
mM HEPES buffer at pH 7.6. For a typical Ki
determination, the Km for ATP in the ATPase activity of
activated p38 reaction is determined in the absence of
15 inhibitor and in the presence of two concentrations of
inhibitor. A stock solution is prepared containing all
of the above components and activated p38 (60 nM) . The
stock solution is aliquotted into vials. A fixed volume
of DMSO or inhibitor in DMSO (final concentration of DMSO
20 in reaction was 2.5%) is introduced to each vial, mixed
and incubated for 15 minutes at room temperature. The
reaction is initiated by adding various concentrations of
ATP and then incubated at 30''c. After 30 minutes, the
reactions are quenched with 50 p.1 of EDTA (0.1 M, final
25 concentration), pH 8.0. The product of p38 ATPase
activity, ADP, is quantified by HPLC analysis.
Separation of ADP from ATP is achieved on a
reversed phase column (Supelcosil, LC-18, 3 ]jm, part no.
5-8985) using a binary solvent gradient of following
30 composition: Solvent A - 0.1 M phosphate buffer
containing 8 mM tetrabutylammonium hydrogen sulfate
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(Sigma Chemical Co., catalogue no. T-7158), Solvent B -
Solvent A with 3 0% methanol.
Ki is determined from the rate data as a
function of inhibitor and ATP concentrations.
5 p38 inhibitors of this invention will inhibit
the ATPase activity of p38.
C. Inhibition of IL-1, TNF, IL-6 and IL-8
Production in LPS-Stimulated PBMCs
10
Inhibitors were serially diluted in DMSO from a
20 mM stock. At least 6 serial dilutions were prepared.
Then 4x inhibitor stocks were prepared by adding 4 p.1 of
an inhibitor dilution to 1 ml of RPMI154 0 medium/10%
15 fetal bovine serum. The 4x inhibitor stocks contained
inhibitor at concentrations of 80 pM, 32 pM, 12.8 pM,
5.12 loM, 2.048 pM, 0.819 pM, 0.328 pM, 0.131 pM, 0,052
pM, 0.021 pM etc. The 4x inhibitor stocks were pre-
warmed at 37°C until use.
20 Fresh human blood buffy cells were separated
from other cells in a Vacutainer CPT from Becton &
Dickinson (containing 4 ml blood and enough DPBS without
Mg^'^/Ca^"^ to fill the tube) by centrif ugation at 1500 x g
for 15 min. Peripheral blood mononuclear cells (PBMCs) ,
25 located on top of the gradient in the Vacutainer, were
removed and washed twice with RPMI164 0 medium/10% fetal
bovine serum. PBMCs were collected by centrif ugation at
500 X g for 10 min. The total cell number was determined
using a Neubauer Cell Chamber and the cells were adjusted
30 to a concentration of 4,8 x 10^ cells/ml in cell culture
medium (RPMI1640 supplemented with 10% fetal bovine
serum) .
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Alternatively;. whole blood containing an anti-
coagulant was used directly in the assay.
100 ^1 of cell suspension or whole blood were
placed in each well of a 96-well cell culture plate.
5 Then 50 lal of the 4x inhibitor stock was added to the
cells. Finally, 50 ]il of a lipopolysaccharide (LPS)
working stock solution (16 ng/ml in cell culture medium)
was added to give a final concentrati on of 4 ng/ml LPS in
the assay. The total assay volume of the vehicle control
10 was also adjusted to 200 ]il by adding 50 ^l cell culture
medium. The PBMC cells or whole blood were then
incubated overnight (for 12-15 hours) at 37° C/5% CO2 in a
humidified atmosphere.
The next day the cells were mixed on a shaker
15 for 3-5 minutes before centrif ugation at 500 x g for 5
minutes. Cell culture supernatants were harvested and
analyzed by ELISA for levels of IL-lp (R&D Systems,
Quantikine kits, #DBL50) , TNF-a (BioSource, #KHC3012),
IL~6 (Endogen, #EH2-IL6) and IL-8 (Endogen, #EH2-IL8)
20 according to the instructions of the manufacturer. The
ELISA data were used to generate dose-response curves
from which IC50 values were derived.
Results for the kinase assay (^^kinase'';
subsection A, above) , IL-1, and TNF in LPS-stimulated
25 PBMC's (^^cell") and IL-1, TNF, and IL-6 in whole blood
(^^WB") for various p38 inhibitors of this invention are
shown in Table 7 below:
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Table 7.
Compound
M.W.
Kinase
IC50 (uM)
Cell IL-1
IC50 (uM)
Cell TNF
ICSO (uM)
WB IL-1
ICSO (uM)
WBTNF
ICSO (uM)
WBIL-6
ICSO (uM)
13
559.55
0.031
0.012
0.022
0.140
0.055
0.083
9
489.43
1.0
0.05
0.05
12.2
20.0
11.0
10
544.51
5.0
2.2
4.3
0.8
Other p38 inhibitors of this invention will
also inhibit phosphorylation of EGF receptor peptide, and
5 will inhibit the production of IL-1, TNF and IL-6, as
well as IL-8, in LPS-stimulated PBMCs or in whole blood.
D. Inhibition of IL-6 and IL-8
Production in IL-l-Stimulated PBMCs
This assay is carried out on PBMCs exactly the
10 same as above except that 50 ]al of an IL-lb working stock
solution (2 ng/ml in cell culture medium) is added to the
assay instead of the (LPS) working stock solution.
Cell culture supernatants are harvested as
described above and analyzed by ELISA for levels of IL-6
15 (Endogen, #EH2-IL6) and IL-8 (Endogen, #EH2-IL8)
according to the instructions of the manufacturer. The
ELISA data are used to generate dose-response curves from
which ICSO values were derived.
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E. Inhibition of LPS-Induced
Prostaglandin Endoperoxide Synthase-2
(PGHS-2, or COX-2) Induction in PBMCs
Human peripheral mononuclear cells (PBMCs) are
5 isolated from fresh human blood buffy coats by
centrifugation in a Vacutainer CPT (Becton & Dickinson) ,
15 X 10 cells are seeded in a 6-well tissue culture dish
containing RPMI 1640 supplemented with 10% fetal bovine
serum, 50U/ml penicillin, 50 p-g/ml streptomycin, and 2 mM
10 L-glutamine. Compounds are added at 0.2, 2.0 and 20 |IM
final concentrations in DMSO. LPS is then added at a
final concentration of 4 ng/ml to induce enzyme
expression. The final culture volume is 10 ml/well.
After overnight incubation at 37°C, 5% C02, the
15 cells are harvested by scraping and subsequent
centrifugation, the supernatant is removed, and the cells
are washed twice in ice-cold DPBS (Dulbecco's phosphate
buffered saline, BioWhittaker ) . The cells are lysed on
ice for 10 min in 50 lal cold lysis buffer (20 mM Tris-
20 HCl, pH 7.2, 150 mM NaCl, 1% Triton-X-100, 1% deoxycholic
acid, 0.1% SDS, 1 mM EDTA, 2% aprotinin (Sigma), 10 jag/ml
pepstatin, 10 |ig/ml leupeptin, 2 mM PMSF, 1 mM
benzamidine, 1 mM DTT) containing 1 ^1 Benzonase (DNAse
from Merck) . The protein concentration of each sample is
25 determined using the BCA assay (Pierce) and bovine serum
albumin as a standard. Then the protein concentration of
each sample is adjusted to 1 mg/ml with cold lysis
buffer. To 100 ]il lysate an equal volume of 2xSDS PAGE
loading buffer is added and the sample is boiled for 5
30 min. Proteins (30 p.g/lane) are size-fractionated on 4-
2 0% SDS PAGE gradient gels (Novex) and subsequently
transferred onto nitrocellulose membrane by
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^lactropJioratic meana ^or 2 lioura at .100 in Towbln
transfer buffer (25 ^ Tri^^ IB2 im glyciae) conteining
20% methariol* After transfer t:he membrane is pr^traated
for 1 tooxir at: room t^mperatiira witJi bio eking biaffer 1 5%
noa-fat dry milk in DPBS supplemented with 0.1% l^mmi-^^Ql
and wshed 3 times in DPBS/0»1% Tiie membrane
ia incubated ot^^eriiight at 4^C witti a 1 ; 2S0 dilution, of
Kioxiocional an t i 2 axitib<^dy (!Fr ana duct io-n
Laboratories j in blocJciixg buffer* After 3 washes in
10 D.PBS/0 .1% lweBri-20jt tlm membrane incubated v/itn a
It 1000 diiutioa of horseradish peroxidase-conji^gated
sheep antisertffii to mouse Ig C&aershain.) ia Jblocfeing biaffer
for 1 E at room temperature* Then the membretie ia i^jaahad
again 3 time-3 in DFBS/0,1?^ f>^eeti--20 , ECL detection
system {Super Signal'^ Cli-^HRP Substrate System^ .Fierce) is
used to determine the levels of ^xpresaioia of C0X.-*2*
2AP70 Inhibition Aasay
20 The activity of BHP 70 is Eteasarad by
detarirdnlBg the phosphorylation poly E4Y {Sigitia
Chemicals ^ St I^qxiIb mq) ^Ith y-^^P AfP (MEM, Boston.^ Ma) *
Reactions are carried out at room temperature in a buffer
contBlning lOQ m mms, pH 7.5, XO mM MgClar 25 him. mcl,
2 5 t JtM DTf and 0.01% BBK. Final coxioer^tratioM of E^70
axxd poly E4Y are 20 nM and 5 pM. respBctiv^ly , test
compQttnds Ib i;mso ffinal concentration of compounds was
30 iM in 1.5% JMSQ} are added to ttie reaction mixture
contaiixing the aboire---descritoed componeats^ ThB reaction
30 is initiated by addition of f-" ATP (final oou.cexit ration
20 pM^ specific acti"Q^ity — 0^018 Ci/maol) > reactioia
allowed to proceed for 12 minutes and then is qumched
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by the addition of 10% TCA containing 200 mM ATP. The
quenched reaction is harvested onto GF/C glass fiber
filter plates (Packard, Meriden, CT) using a Tomtec 9600
cell harvester (Tomtec, Hamden, CT) . The plates are
5 washed with 5% TCA containing 1 mM ATP and water. 50 v^l
of scintillation fluid is added to the plates, which are
then counted using a Packard scintillation counter
(Packard, Meriden, CT) . IC50 values for inhibitory
compounds were determined using the same assay at a
10 series of compound concentrations.
While we have hereinbefore presented a number
of embodiments of this invention, it is apparent that our
basic construction can be altered to provide other
15 embodiments which utilize the methods of this invention.
20
25
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CLAIMS
We claim:
1. A compound of the formula
(la) ,
(lb) ,
O
(Ic)
or (Id) ,
wherein each of Qi and Qz are independently
selected from a phenyl or 5-6 membered aromatic
heterocyclic ring system, or a 8-10 membered bicyclic
ring system comprising aromatic carbocyclic rings,
aromatic heterocyclic rings or a combination of an
aromatic carbocyclic ring and an aromatic heterocyclic
ring;
wherein the rings that make up Qi are
substituted with 1 to 4 substituents , each of which is
independently selected from halo; C1-C3 alkyl optionally
substituted with NR'2/ OR', CO2R' or CONR'2; O- {C1-C3) -
alkyl optionally substituted with NR'2. OR', CO2R' or
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CONR'2; NR'2; OCF3; CF3/ NO2; CO2R'; CONR'; SR';
S(02)N(RM2; SCF3; CN; N(R')C(0)R'; N(R' )C(0)OR^•
N(R' )C(0)C(0)R'; N(R' ) s (02)R^- n(r')r\' n(r^)2; or';
oc(0)rS- op(o)3H2; or n=c-n(r' ) 2;
wherein the rings that make up Q2 are
optionally substituted with up to 4 substituents, each of
which is independently selected from halogen; C1-C3
straight or branched alkyl optionally substituted with
R', NR'2. OR', CO2R', S(02)N(R')2a N=C-N(R')2, R^ O-
P(03)H2, or CONR'2; O- (C1-C3) -alkyl; O- (C1-C3) -alkyl
optionally substituted with NR'2 A OR', CO2R' , S(02)N{R')2,
N=CR'-N(R' ) 2, R^/ OP(03)H2, or CONR'2; NR'2; OCF3; CF3; NO2;
CO2R'; CONR'2; R^; OR^; NR^; SR^; C(0)R^; C(0)N(R')R^;
C{0)OR^; SR'; S(02)N(R')2; SCF3; N=CR'-N(R' )2; or'; O-CO2R';
N(R')C(0)R'; N(R' )C(0)0RS- N(R' )C(0)C(0)R'; N(R')S(02)R^
N(R')R'; N(R')2; ORS- 0C(0)R'; 0P(0)3H2; K; or CN;
wherein each R' is independently selected from
hydrogen; (C1-C3) -alkyl ; (C2-C3) -alkenyl or alkynyl;
phenyl or phenyl substituted with 1 to 3 substituents
independently selected from halo, methoxy, cyano, nitro,
amino, hydroxy, methyl or ethyl; or a 5-6 membered
heterocyclic ring system optionally substituted with 1 to
3 substituents independently selected from halo, methoxy,
cyano, nitro, amino, hydroxy, methyl or ethyl;
wherein each R is independently selected from
hydrogen, -R^, -N(R^)2, -0R^ SR^, -C (O) -N (R^) 2, -S(02)-
N{R^)2, -C{0)-0R^ or -C(0)R^ wherein two adjacent R are
optionally bound to one another and, together with each Y
to which they are respectively bound, form a 4-8 membered
carbocyclic or heterocyclic ring;
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wherein each R is independently selected from
hydrogen; or (C1-C3) -alkyl or (C1-C3) -alkenyl, each
optionally substituted with -N(R')2^ -OR', SR' , -0-C (O) -
N(R')2, -C(0)-N{R' )2/ -S (O2) -N(R' )2, -C(0)-OR', -NSOaR^ -
NSOzR^, -C (O)N(R' ) (R^) , -NC(0)R^ -N(R')(R^), -N{R')(R^), -
C{0)R^, -C(0)N(R' ) (R^) , -N(R^)2, -C (O) N=C (NH) 2 orR^;
3
wherein each R is independently selected from
5-8 menibered aromatic or non-aromatic carbocyclic or
heterocyclic ring systems each optionally substituted
with R' , R^ -C(0)R', -C(0)R^ -C(0)OR^ or -K; or an 8-10
membered bicyclic ring system comprising aromatic
carbocyclic rings, aromatic heterocyclic rings or a
combination of an aromatic carbocyclic ring and an
aromatic heterocyclic ring each optionally substituted
with R^, R^, -C(0)R', -C(0)R^, -C (O) OR^ or -K;
4
wherein each R is independently selected from
R'; (Ci"C7) -straight or branched alkyl optionally
substituted with R' , N(R')2/ OR', CO2R' , C0N(R')2/
S02N{R')2or S02N(R^)2; or a 5-6 membered carbocyclic or
heterocyclic ring system optionally substituted with
N(R')2, OR', CO2R', C0N(R')2, S02N(RM2 or S02N(R^)2;
wherein each R is independently selected from
hydrogen, (C1-C3) -alkyl, or (C1-C3) -alkenyl ; each
optionally substituted with -N(R')2, -OR', SR' , -C (O) -
N(R')2, -S (O2) -N(R' )2, -C(0)-OR', -N-S (O2 ) (R' ) , -NS02R^, -
C(0)N(R' ) (R^) , -NC(0)R', -N(R')(R^), -C(0)R^, -
C(0)N=C{NH)2 or R^;
wherein each R is independently selected from
5-8 membered aromatic or non-aromatic carbocyclic or
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heterocyclic ring systems each optionally substituted
with R' , -C(0)R' or -C(0)OR'; or an 8-10 meinbered
bicyclic ring system comprising aromatic carbocyclic
rings, aromatic heterocyclic rings or a combination of an
aromatic carbocyclic ring and an aromatic heterocyclic
ring each optionally substituted with R' , -C(0)R' or
C (O) OR' ;
wherein R is selected from H, halogen, or a
(C1-C3) straight chain or branched alkyl;
wherein ach Y is independently selected from N
or C. If either Y is N, then R or U attached to Y is a
lone pair of electrons;
wherein Z is CH, N, C(0CH3), C(CH3), C(NH2),
C (OH) or C (F) ;
wherein each U is independently selected from R
or J;
wherein each J is independently selected from a
(C1-C4) straight chain or branched alkyl derivative
substituted with T;
wherein each T is independently selected from
either O (V) or N{H) (V) ;
wherein each V is independently selected from
C(0)N=C(R) {N(R)2)/ wherein the two geminal R on the
nitrogen are optionally bound to one another to form a 4-
8 membered carbocyclic or heterocyclic ring;
wherein each K is independently selected from a
(C1-C4) straight chain or branched alkyl derivative
substituted with D, or "0P(0) (OH) 2;
wherein each D is independently selected from
either enantiomer of
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PCT/USOl/25015
wherein each M is iudependentiy selected from
either O or NH;
wherein each G is independently selected from
NH2/ OH, or H;
wherein each Re is independently selected from
OH^ C(0)OH^ (CX-C7) -straight or branched alkyl
optionally substituted with N(RM2/ OR^ / C02R^ r C0N(RM2/
or S02N(R )2r or a 5-6 metubered carbocyclic, heterocyclic
or heteroaryl ring system optionally substituted with
N(R')2/ 0R% CO2RS CON{RM2/ or S02N(R^)2; wherein G and
Rb are optionally bound to one another to form a ring.
2* The compound according to claixn 1^ wherein
Qi ±B selected from phenyl or pyridyl containing 1 to 3
substituents independently selected from chloro/ fluoro,
bromo, -OH, -0(CH2)2CH3.
3, 4-methylenedioxy, -NfCHs)^/ -NH^S (O) 2-phenyl, -HH-C{0)0-
CH2-4"pyridiner (O) CHa^taorpholine/
-im-C (O) CH2-N (CH3) 2/ -NH-C (O) CHa-piperazine,
-NH~C (O) CH2"pyrrolidine^ -NH-C (O) C (O) -morpholine^
-mi-C (O) C (O) -piperazine, --^NH-C (O) C (O) --pyrrolidine/
"O-CCOlCHa-NCCHala. or --O-- {CH2J a^N {CH3) 2 and wherein at
least one of said siobstituents is in the ortho position.
3* The compound according to claim 2, wherein
Qi contains at least two substituentS/ both of which are
in the ortho position.
4, The compound according to claiin 2, wherein
Qi is selected from:
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OCH3
OCH3
OCH3
^ HsCCTT^OCHs
NH2
'^^^ H3CO
OCH3
H3
H3<rN^CH
3 C
CI
CrV^OCHs HaCV^OCH
H3C
NH2
.NH2
H3I
CI
CI
OCH3
HsCV^^OCH;
CI
■OCH3
NH2
OH
.NH2
-OH
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f or
5. The compound according to claim 4, wherein
Qi is selected from 2-f luoro-6-trif luoromethylphenyl,
2, 6-dif luorophenyl, 2, 6-dichlorophenyl;, 2-chloro-4-
hydroxyphenyl, 2-chloro-4-aminophenyl, 2, 6"dichloro-4-
aminophenyl, 2, 6-dichloro-3-aminophenyl, 2, 6-dimethyl-4-
hydroxyphenyl, 2-methoxy-3^ 5-dichloro-4-pyridyl, 2-
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chloro-4,5 methyl enedioxy phenyl, or 2-chloro-4- (N-2-
morpholino-acetamido) phenyl .
6. The compound according to claim 1^ wherein
Qz is selected from phenyl, pyridyl or naphthyl and
wherein Q2 optionally contains up to 3 substituents, each
of which is independently selected from chloro, fluoro,
bromo, methyl, ethyl, isopropyl, -OCH3, -OH, -NH2, -CF3, -
OCF3, -SCH3, -OCH3, -C(0)OH, -C(0)0CH3, -CH2NH2, -N(CH3)2,
-CH2-pyrrolidine and -CH2OH.
7, The compound according to claim 6, wherein
Q2 is selected from:
OH
O2H
OCH3
•SCH:
SCH
■CH-
■CH3
OCH-
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73
N(CH3)2
Ui
rt
^NH
CI
OH
OH
NH
CI
f J
C!
HaW^H
CO2H
CI
CI
bo2H
■NH
\ /
L
HN^N
■NH
HhT^N
^ <- f S
OH
CH3
Sdh
OH
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\aiisubstltuted or uasubstitiated phenyls
8^ The Qompomxd according to claim 7^ whe*
Qo is selected from phenyl^. Z-tsopxopylphBnylt 3/4-
dimethylphenyl/ 2--at"iiylp}ienyl ^ S-^f luorophanylr 2--
carbomethox.:ylph^iiyl ^ 2^carbaKirpiienyl ^ 2--imtbyl-4-
tueth^^l eii0hy<iroxyphenyl^ 4-- f luoropheriyl^ 2 --methyl-- 4 ---^
fluorophenylr 2'-chloro^4-f luorphenyl/ 2^ 4--difluorophanyl^
2-^hydxo.xy*-4 - f luo^rpheiiy 1 or 2 "-KtBthyletxBhydroKy- 4 -
f luoropiiens'^i/ l-^Baphthyl;. 3-chloro**2"-Biet]:iy^lettehydrox-y, So-
cial oro--' 2 --methyl f or 4-*-f *
9* Th.B corapaund accordirtg to claim wherein
10* ThB compound according to claim 9^ wherein
tfaa K attached to Y is independently selected from
iiydrogexi or methyl -
11 » "fhe compoimd according to clam if i^^hereixi
J a atoiE chain te.minating in an ^Icoholi amine^
carboxylic acld^ ester a amide, ^ amldirie or lieterocycle
12* Tile compound according to claim li^
where ixi J selected from:
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o
N
O
O
O N
N
O
N
NH
I or
13. The compound according to claim 1 wherein
K is selected from:
NH2
O
O' OH
o
O
NH2
O
NH2
or
14. The compound according to claim 1^ wherein
the compound is selected from any one of the compounds
depicted in Tables 1-3.
15. The compound according to claim \, wherein
the compound is
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17. The compound according to claim 1, wherein
the compound is
18. The compound according to claim 1, wherein
the compound is
19. The compound according to claim 1^. wherein
the compound is
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20. A pharmaceutical composition comprising an
amount of a compound according to any one of claims 1 to
19 effective to inhibit p38, and a pharmaceutically
acceptable earner •
21. A method of treating or preventing
inflammatory diseases, autoimmune diseases, destructive
bone disorders, proliferative disorders, infectious
diseases, neurodegenerative diseases, allergies,
reperfusion/ischemia in stroke, heart attacks, angiogenic
disorders, organ hypoxia, vascular hyperplasia, cardiac
hypertrophy, thrombin-induced platelet aggregation or
conditions associated with prostaglandin endoperoxidase
synthase-2 in a patient, said method comprising
administering to said patient a composition according to
claim 20.
22. The method according to claim 21, wherein
said method is used to treat or prevent an inflammatory
disease selected from acute pancreatitis, chronic
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pancreatitis, asthma, allergies, or adult respiratory
distress syndrome ,
23. The method according to claim 21, wherein
said method" is used to treat or prevent an autoimmune
disease selected from glomerulonephritis, rheumatoid
arthritis, .systemic lupus erythematosus, scleroderma,
chronic thyroiditis. Graves' disease, autoimmune
gastritis, diabetes, autoimmune hemolytic anemia,
autoimmune neutropenia, thrombocytopenia, atopic
dermatitis, chronic active hepatitis, myasthenia gravis,
multiple sclerosis, inflammatory bowel disease,
ulcerative colitis, Crohn's disease, psoriasis, or graft
vs. host disease.
— 2-4,- - The- method- -according to Glaim™2X,~-wherean_
said method is used to treat or prevent a destructive
bone disorders selected from osteoarthritis, osteoporosis
or multiple myeloma-related bone disorder.
25- The method according to claim 21, wherein
said method is used to treat or prevent a proliferative
disease selected from acute myelogenous leukemia, chronic
myelogenous leukemia, metastatic melanoma, Kaposi's
sarcoma, or multiple myeloma.
26. The method according to claim 21, wherein
said method is used to treat or prevent an infectious
disease selected from sepsis, septic shock, or
Shigellosis ,
SUBSTITUTE SHEET (RULE 26)
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27. The method according to claim 21, wherein
said method is used to treat or prevent a viral disease
selected from acute hepatitis infection, HIV infection or
CMV retinitis.
28. The method according to claim 21, wherein
said method is used to treat or prevent a
neurodegenerative disease selected from Alzheimer' s
disease, Parkinson's disease, cerebral ischemia or
neurodegenerative disease caused by traumatic injury.
29. The method according to claim 21, wherein
said method is used to treat or prevent
ischemia/reperf usion in stroke or myocardial ischemia,
— renal ischemia-/ -heart attacks, organ-hypoxia or thrombin-
induced platelet aggregation.
30. The method according to claim 21, wherein
said method is used to treat or prevent a condition
associated with prostaglandin endoperoxide synthase-2
selected from edema, fever, analgesia or pain.
31. The method according to claim 30, wherein
said pain is selected from neuromuscular pain, headache,
cancer pain, dental pain or arthritis pain.
32. The method according to claim 21, wherein
said method is used to treat or prevent an angiogenic
disorder selected from solid tumors, ocular
neovasculization, or infantile haemangiomas .
SUBSTITUTE SHEET (RULE 26)
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-Bi-
as . A method of treating or preventing a p38-
raediated disease, said method comprising administering to
said patient a composition according to claim 20.
34. A pharmaceutical composition comprising an
amount of a compound according to any one of claims 1 to
19 effective to inhibit ZAP70, and a pharmaceutically
acceptable carrier .
35. A method of treating or preventing organ
or tissue rejection associated with transplantation,
autoimune disease, cancer, multiple sclerosis, graft
versus host disease, and Kawasaki syndrome, said method
comprising administering to said patient a composition
according to claim 34 .
36. The method according to claim 35, wherein
said method is used to treat or prevent an autoimmune
disease selected from rheumatoid arthritis, systemic
lupus erythematosus (SLE) , psoriasis, Sjogren's Syndrome,
thyroiditis, pulmonary fibrosis, bronchiolitis
obliterans, hemolytic anemia and Wegener's
granulomatosis .
37. The method according to claim 35, wherein
said method is used to treat or prevent a cancer selected
from leukemia and lymphoma.
38. A method of treating or preventing a
ZAP7 0-mediated disease, said method comprising
administering to said patient a composition according to
claim 34.
SUBSTITUTE SHEET (RULE 26)
INTEBNATIONAL SEARCH REPORT
Interna 1 Application No
PCT/Ub 01/25015
A. CLASSIFICATION OF SUBJECT MATTER , , , /
IPC 7 C07D213/75 C07D401/12 A61K31/4418 A61P7/00 A61P9/00
A61P17/00 A61P25/00 A61P31/00 C07F9/58
According to international Patent Classification (IPC) or to boXh national ciassification and IPC
B. FIELDS SEARCHED
Minimunn documentation searcbied (classification system foliowed by classification symbols)
IPC 7 C07D A61K A61P C07F
Documentation searched other than minimum documentation to the e)ctent that such documents are included in the fields searched
Electronic data base consulted during the internatfonal search (name of data base and, where pracltcal, search terms used)
EPO-Internal , CHEM ABS Data
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Citation of document, with Indication, where appropriate, of the relevant passages
Relevant to claim No.
X
wo 99 58502 A (BELLON STEVEN ;BEMIS GUY
(US); VERTEX PHARMA (US); COCHRAN JOHN (U)
18 November 1999 (1999-11-18)
cited in the application
page 71, Hne 17 - line 20; claims;
examples
1-40
E
WO 01 70695 A (EVINDAR GHOTAS ;BEMIS GUY
(US); VERTEX PHARMA (US); SALITURO FRANC)
27 September 2001 (2001-09-27)
claims 1,8,11-23
1,13,14,
18,20-40
1 j Further documents are listed in the continuation of box C.
|)( j Patent famiiy members are listed in annex.
° Special categories of cited documents ;
'A' document defining the general state of the art which is not
considered to be of particular relevance
'E' earlier document but published on or afterthe international
filing date
"L" document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition or
other means
'P' document published prior to the international filing date but
later than the priority date claimed
'T* later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention
'X' document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document Is taken alone
"Y" document of particular relevance; the claimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.
document member of the same patent family
Date of the actual completion of the International search
Date of maiing of the International search report
22 January 2002
04/02/2002
Name and mailing address of the ISA
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl,
Fax; (+31-70)340-3016
Authorized officer
Bosma,
P
Form PCT/lSA/210 (second sheet) (July 1992)
International Application No. PCTAJS 01 y^5015
FURTHER INFORMATION CONTINUED FROM PCT/JSA/ 210
Continuation of Box 1.2
Claims Nos.: 1-13, 20-40 (partially)
Present claims 1-13 relate to an extremely large number of possible
compounds. Support within the meaning of Article 6 POT and/or disclosure
within the meaning of Article 5 POT is to be found, however, for only a
very small proportion of the compounds claimed. In the present case, the
claims so lack support, and the application so lacks disclosure, that a
meaningful search over the whole of the claimed scope is impossible.
Consequently, the search has been carried out for those parts of the
claims which appear to be supported and disclosed, namely those parts
relating to the examples and to the compounds according to claims 1-13 of
the present formulae (la), (lb), (Ic), (Id) in which Ql has the meanings
as defined in claim 4, and Q2 has the meanings as defined in claim 7, and
also to the use of these compounds according to claims 20-40.
The applicant's attention is drawn to the fact that claims, or parts of
claims, relating to inventions in respect of which no international
search report has been established need not be the subject of an
international preliminary examination (Rule 66.1(e) POT). The applicant
is advised that the EPO policy when acting as an International
Preliminary Examining Authority is normally not to carry out a
preliminary examination on matter which has not been searched. This is
the case irrespective of whether or not the claims are amended following
receipt of the search report or during any Chapter II procedure.
INTERNATIONAL SEARCH REPORT
nation on patent family members
Patent document
cited in search report
Publication
date
Interns Application No
PCT/u:i 01/25015
WO 9958502
A
18-11-1999
Patent farriliy
member(s)
Publication
date
AU
3792399
A
29-11-1999
BG
105031
A
31-10-2001
BR
9911786
A
03-04-2001
CN
1306512
T
01-08-2001
EP
1077943
Al
28-02-2001
NO
20005673
A
10-01-2001
PL
344046
Al
24-09-2001
SK
16882000
A3
10-05-2001
WO
9958502
Al
18-11-1999
UO 0170695 A 27-09-2001 WO 0170695 Al 27-09-2001
Form PCT/lSA/210 (paienl family annex) (July 1992}
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